Although human B cell hybridomas secreting Ig of defined specificity have been produced in several laboratories, the technology in this area is well behind that available in animal systems. Difficulties still exist in identifying optimal parental fusion partners, enriching appropriate antigen-activated nomal B cells prior to fusion, and growing stable hybridomas after fusion. This study will attempt to improve these technological issues while addressing several basic questions about human hybridoma formation and human monoclonal antibodies. Attempts will be made to produce new human mutant B lineage cell lines for use as parental fusion partners. This will be done by deriving human lines from B cells at all stages of differentition from pre-B cells to plasma cells and then mutating these lines to produce suitable variants. Non-secreting and non-synthesizing cell lines will be sought in an analogous fashion. We will extend our previous observations that Epstein-Barr virus transformed cells act effectively as parental fusion lines by comparing EBV-negative myeloma lines with those derived by EBV infection for fusion efficiency, stability and Ig production. These lines will be fused with in vivo antigen activated normal B cells from individuals immunized either intentionally or by natural exposure which will be isolated by cell sorting using murine monoclonal antibodies to human leukocyte activation antigens. In addition attempts will be made to define optimal conditions for in vitro immunization of B lymphocytes. Cocultures of normal peripheral blood or tonsillar mononuclear cells with antigen plus either allogeneic T cells, or low doses of various polyclonal B cell activators, or with various T cell derived B cell growth and differentiation factors. Certain stable antigen-sepcific human B cell hybridomas will be used as monoclonal models of normal human B cell physiology. These immortalized cells will be used to study B cell activation, differentiation and Ig isotype class switching. The studies proposed above will provide valuable information regarding human B cell hybridization and the uses of human B cell hybridoma and the resultant human monoclonal antibodies for the diagnosis and therapy of human diseases.